exam 3 imm gen quiz

phosphorylation: kinases

Receptor tyrosine kinases (RTKs) contain kinase domain

steps of phosphorylation

1. the kinase domain is an intrinsic part of the receptor
2. ligand binding dimerizes the receptor activating the kinases which phosphorylate each other
3. the activated kinases phosphorylate downstream substrates

scaffold proteins

organize groups of intracellular signaling molecules into signaling complexes. recruit multiple proteins

adaptor proteins

link two or more proteins together. examples: SH2: p-Tyr, SH3: proline rich, PH: phosphoinositides

small G proteins/ GTPases

Ras, Rac, Rho, Cdc42

role of small G proteins

hydrolyze GTP into GDP and Pi. GTP is active, GDP is inactive

GEFs and GAPs

regulate small G protein activity. high amounts of GEF, low amounts of GAP

steps for G proteins switching signals on and off

1.in the resting state, small G proteins are bound to GDP and are inactive
2. signaling activates GEFs such as Sos which increase the rate of exchange of GDP for GTP
3. the GTP bound small G protein is the active effector molecule
4. overtime the small G protein hydrolyzes the GTP to GDP and becomes inactive. this process is accelerated by GAPs

recruitment of proteins to the plasma membrane

1. binding to phosphorylated sites on a membrane associated protein
2. recognition of activated small G proteins
3. PI 3 kinase phosphorylates PIP2 to generate PIP3
4. binding to membrane lipids

Post translational modification activates and terminates signaling

1. dephosphorylation of phosphorylated substrates
2. ubiquitin mediated degradation by proteasome
3. ubitiquin mediated degradation in the lysosome

amplification of initial signals: kinase cascades

1. amplification by kinase cascades
2. signaling results in the release of the second messenger calcium
3. calcium rapidly diffuses throughout the cell and induces conformational changes in calmodulin

T-cell receptor complex

- variable antigen binding chains (binds to peptide MHC complex)
- invariable chains stabilize a:B heterodimer
- CD3 complex: CD3y
- contains ITAMs in their intracellular regions that can be phosphorylated

B-cell Receptor

- variable antigen binding chains
- H:L heterotetramer
- VDJ recombination
- binds to epitopes on native antigens
- invariable chains: Iga:Igb, two ITAMs

T cell receptors and co receptors

CD8 and MHC1, CD4 and MHC 2

aggregation

during presentation, many TCRs are bound to MHC peptide complexes, possible microclustering

conformational changes

changes within the complex: expose the e and z chains from the membrane leads to the phosphorylation of ITAMs

Transduce signal to initiate intracellular signaling

Lck phosphorylates the ITAMs in the TCR upon co-receptor engagement with antigen MHC. ZAP-70 is recruited by tandem SH2 domains to the ITAMS and is phosphorylated by Lck

Engagement of co-receptors

1. kinase Lck phosphorylates ITAMs and recruits kinase ZAP70 through SH2 domains
2. Lck phosphorylates ZAP70

Lck

Src family tyrosine kinase, constitutively associated with CD4 and CD8

ZAP70

zeta chain associated protein of 70 kDa; tyrosine kinase; dual SH2 domains

Lck activity is regulated by

phosphorylation and dephosphorylation through CD45, Csk. Full Lck activation requires autophosphorylation

ZAP70 Phosphorylates Scaffold proteins

1. kinase Lck phosphorylates ITAMs and recruit kinase Zap70 through SH2 domains
2. Lck phosphorylates Zap70 and activates it
3. activated Zap70 phosphorylates LAT and SLP76
4. phosphorylated LAT recruits Gads and slp 76

LAT

linker for activation of T cells; transmembrane scaffold

SLP-76

SH2 domain containing leukocyte protein of 76 kDa; cytoskeleton rearrangement

Gads

GRB2-related adaptor molecule; constitutively associated with SLP76

Recruitment and activation of Plc-y (cont from zap70)

5. slp-76 associates with other molecules leading to cytoskeletal rearrangement
6. LAT: Gads:Slp76 complex recruits phospholipase Cy (PLCy)
7. phosphorylated SLP 76 also recruits and activates Itk
8. ITk phosphorylates and activates PLC-y

activation of PLC-y generates second messengers

PLC-y generates two second messengers, leading to three responses: transcription activation, metabolism upregulation, and cytoskeleton rearrangement and increased adhesion. PLC-y hydrolyzes PIP2 into DAG and IP3

PLC-y hydrolyzes PIP2 into

DAG and IP3

activation of NFAT upon Calcium entry

phosphorylation on serine and threonine residues keeps NFAT in the cytoplasm of unstimulated cells. calcium entry activates the serine/threonine phosphatase calcineurin which dephosphorylates NFAT. Dephosphorylated NFAT enters the nucleus and activates gene transcription

NFAT

nuclear factor of activated T cells; stays in cytoplasm of unstimulated cells

Ras

small GTPases; GTP bound (active)/ GDP bound (inactive)

RasGRP

recruited by DAG

MAPK

mitogen activated protein kinase

Elk-1

transcription factor

Ras Activation Stimulates MAPK cascade

1. Ras is initially inactive, TCR signaling produces DAG which recruits RasGRP to the membrane where it activates RAs
2. Ras activates Raf which phosphorylates Mek, which phosphorylates Erk
3. activated Erk enters the nucleus and activates transcription factors such as Elk-1

protein kinase C activates NF-kb and Ap-1

DAG recruits protein kinase C 0 to the plasma membrane PKC phosphorylates CARMA1 and then leads to the activation of Nf-kb and Ap1

PIP3 recruits PDK1 and Akt to cell membrane

leads to PDK1 phosphorylate and activate Akt